The full gene sequence encoding for the Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase (PKA) regulatory (R) subunits was cloned. A poly-His tagged construct was generated [TeqR-like(His)₈], and the protein was expressed in bacteria and purified to homogeneity. The size of the purified TeqR-like(His)₈ was determined to be ∼57,000 Da by molecular exclusion chromatography indicating that the parasite protein is a monomer. Limited proteolysis with various proteases showed that the T. equiperdum R-like protein possesses a hinge region very susceptible to proteolysis. The recombinant TeqR-like(His)₈ did not bind either [³H] cAMP or [³H] cGMP up to concentrations of 0.40 and 0.65 μM, respectively, and neither the parasite protein nor its proteolytically generated carboxy-terminal large fragments were capable of binding to a cAMP-Sepharose affinity column. Bioinformatics analyses predicted that the carboxy-terminal region of the trypanosomal R-like protein appears to fold similarly to the analogous region of all known PKA R subunits. However, the protein amino-terminal portion seems to be unrelated and shows homology with proteins that contained Leu-rich repeats, a folding motif that is particularly appropriate for protein-protein interactions. In addition, the three-dimensional structure of the T. equiperdum protein was modeled using the crystal structure of the bovine PKA R^Iα subunit as template. Molecular docking experiments predicted critical changes in the environment of the two putative nucleotide binding clefts of the parasite protein, and the resulting binding energy differences support the lack of cyclic nucleotide binding in the trypanosomal R-like protein.

克隆了编码cAMP依赖性蛋白激酶（PKA）调节（R）亚基的锥虫锥虫直系同源基因的完整基因序列。产生了一个多组氨酸标签的构建体[TeqR-like（His）₈ ]，该蛋白在细菌中表达并纯化至同质。通过分子排阻色谱法测定纯化的TeqR-like（His）₈的大小约为57,000 Da，表明该寄生虫蛋白是单体。用各种蛋白酶进行的有限的蛋白水解表明，T。equiperdum R-like蛋白具有一个非常易于蛋白水解的铰链区。重组TeqR样（His）₈不结合[ ³ H] cAMP或[ ³H] cGMP分别达到0.40和0.65μM的浓度，并且该寄生虫蛋白或其蛋白水解生成的羧基末端大片段均无法与cAMP-Sepharose亲和柱结合。生物信息学分析预测，锥虫R样蛋白的羧基末端区域似乎与所有已知PKA R亚基的类似区域折叠相似。然而，蛋白质的氨基末端部分似乎无关，并显示出与含有富亮氨酸重复序列的蛋白质的同源性，该序列是特别适合蛋白质与蛋白质相互作用的折叠基序。此外，使用牛PKA R ^I的晶体结构对木贼锥虫蛋白质的三维结构进行了建模。以α亚基为模板。分子对接实验预测了寄生虫蛋白两个假定的核苷酸结合裂隙的环境中的关键变化，并且由此产生的结合能差异支持锥虫R样蛋白中环状核苷酸结合的缺乏。